Yeast Expressing a Phage Endolysin Reduces Endogenous Clostridium perfringens Ex Vivo in 21-Day-Old Broiler Chicken Intestinal Fluids.

The phage endolysin PlyCP41 when purified from Escherichia coli exhibits lytic activity against Clostridium perfringens (CP) in vitro. The anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast (Saccharomyces cerevisiae) was verified in phosphate buffered saline via mixing experiments with cultured CP and transgenic yeast slurries followed by serial dilution plating and colony counts on tryptose sulfite cycloserine (CP indicator) plates. The transgenic yeast containing PlyCP41 resulted in a log10 4.5 reduction (99.997%; P < 0.01) of the cultured CP. In addition, this serial dilution plating assay was used to demonstrate that transgenic yeast slurries could reduce the endogenous CP content in fluids from three different gastrointestinal regions (proximal, medial, and distal) from 21-day-old broiler chickens. The transgenic yeast treatment of gut slurries resulted in a log 10 1.19, 4.53, and 1.28 reduction in proximal, medial, and distal gut slurries (90% to 99.99% of the endogenous CP; P < 0.01), respectively, compared to nontreatment controls. These results indicate that the phage endolysin PlyCP41 expressed in S. cerevisiae is effective at reducing the endogenous CP in gastrointestinal fluids of broiler chickens. Future studies will measure the anti-CP effect in vivo by administering transgenic yeast to broiler chickens in the feed.

Levadura que expresa una fago-endolisina reduce la presencia endógena de Clostridium perfringens Ex vivo en fluidos intestinales de pollos de engorde de 21 días. La fago endolisina PlyCP41, cuando se purifica a partir de Escherichia coli, exhibe actividad lítica contra Clostridium perfringens (Cp) in vitro. La actividad anticlostridial de la endolisina PlyCP41 expresada en levadura transgénica (Saccharomyces cerevisiae) se verificó en solución salina amortiguada con fosfato mediante experimentos de mezclas con cultivos de C. perfringens y suspensiones de levadura transgénica, seguido de cultivos de diluciones en serie y recuentos de colonias en placas de triptosa sulfito cicloserina (TSC; indicador para C. perfringens). La levadura transgénica que contenía PlyCP41 dio como resultado una reducción de log10 4.5 (99.997%; P <0.01) en el cultivo de C. perfringens. Además, este ensayo de dilución en serie en placas se utilizó para demostrar que las suspensiones de levadura transgénica podrían reducir el contenido de C. perfringens endógeno en fluidos de tres regiones gastrointestinales diferentes (proximal, medial y distal) de pollos de engorde de 21 días de edad. El tratamiento con levadura transgénica de las suspensiones intestinales dio como resultado una reducción de log10 de 1.19, 4.53 y 1.28 en las suspensiones intestinales proximal, medial y distal (90% a 99.99 % de C. perfringens endógena; P < 0.01), respectivamente, en comparación con los controles no tratados. Estos resultados indican que la fago-endolisina PlyCP41 expresada en S. cerevisiae es eficaz para reducir el contenido endógeno de C. perfringens en los fluidos gastrointestinales de pollos de engorde. Los estudios futuros medirán el efecto contra C. perfringens in vivo mediante la administración de levadura transgénica a pollos de engorde en el alimento.

Clostridium and Cryptosporidium outbreak linked to a splash pad.

BACKGROUND: . Splash pads for recreational purposes are widespread. Using these pads can pose a health risk if they lack installation regulation and water quality supervision. Our aim was to describe a waterborne disease outbreak caused by Clostridium perfringens and Cryptosporidium spp. in a Barcelona district and the measures taken for its control. METHODS: . On August 2018, 71 cases of acute gastroenteritis were detected, affecting people who used a splash pad or were in contact with a user. Microbiological and environmental investigations were carried out. A descriptive analysis of the sample and Poisson regression models adjusted for age and sex were performed, obtaining frequencies, median values, and adjusted prevalence ratios with their 95% confidence intervals. RESULTS: The median age of the cases was 6.7 years, 27 (38%) required medical care, and three (4.2%) were hospitalized. The greater the number of times a person entered the area, the greater the number of symptoms and their severity. Nineteen (76%) of the 25 stool samples collected from cases showed the presence of one or both pathogens. Environmental investigations showed deficiencies in the facilities and identified the presence of both species in the splash pad. Health education and hygiene measures were carried out, and 14 days after the closure of the facilities, no more cases related to the pad were recorded. CONCLUSIONS: . Specific regulations are needed on the use of splash pads for recreational purposes. Until these regulations are in place, these types of facility should comply with the regulations that apply to swimming pools and spas, including those related to the design of the tanks, water recirculation systems, and adequate disinfection systems.

Evaluation of long-term supplementation of a Bacillus subtilis direct-fed microbial and enzymatically hydrolyzed yeast cell culture product used alone or in combination on Clostridia, Clostridium perfringens, Escherichia coli, and Salmonella prevalence in beef steers.

The objective was to determine the influence of long-term supplementation (258 d) of a direct-fed microbial (DFM) and/or yeast cell wall (YCW) product on bacterial populations in beef steers. Single-sourced Charolais × Red Angus steers (n = 256; body weight = 246 ±â€…1.68 kg) were used in a randomized complete block design and blocked by location into one of four treatments: 1) fed no DFM and no YCW (Control); 2) fed only the DFM (DFM; Certillus CP B1801 Dry, 28 g/steer d-1 ); 3) fed only the YCW (YCW; Celmanax; 18 g/steer d-1 ); and 4) fed the DFM and the YCW (DFM+YCW). Steers were vaccinated for respiratory and clostridial diseases and treated for internal and external parasites at processing and individually weighed on days 1, 14, 42, 77, 105, 133, 161, 182, 230, and 258. To determine bacterial prevalence, fecal samples were collected on days 1, 14, 77, 133, 182, and 230 and environmental (pen area, feed, and water) samples were collected at the beginning of the week when cattle were weighed. No treatment × day interactions or treatment effects (P > 0.05) were observed between treatment groups at any sampling days for the bacterial populations. Samples on days 1, 133, and 182 had greater (P < 0.05) Clostridia levels compared to the other sampling points but were not different from each other. Clostridia levels were also greater (P < 0.05) on day 77 compared to days 14 and 230. Samples on days 77 and 230 had greater (P < 0.05) Clostridium perfringens levels compared to the other sampling points but were not different (P > 0.05) from each other. Samples on days 1 and 14 had lower (P < 0.05) total Escherichia coli levels compared to the other sampling points but were not different (P > 0.05) from each other. Escherichia coli levels on day 77 were higher (P < 0.05) compared to days 133, 182, and 230. Little Salmonella prevalence (1.5%) was observed throughout the study. This study had greater levels of Clostridia compared to small and large commercial feedlots in the Church and Dwight research database, but C. perfringens, total and pathogenic E. coli, and Salmonella prevalence were notably lower. Collectively, there were no appreciable treatment influences on bacterial populations. These data further indicate a low pathogenic bacterial challenge at the trial site, which could partially explain the lack of differences with DFM or YCW supplementation. The DFM and YCW used alone or in combination cannot be expected to show additional benefits when animals are relatively unstressed with a low pathogenic bacterial challenge.

The objective of this research was to determine the influence of long-term supplementation (258 d) of a direct-fed microbial (DFM) and/or yeast cell wall (YCW) product on bacterial populations in beef steers. Collectively, there were no appreciable treatment influences on bacterial populations. These data further indicate a low pathogenic bacterial challenge at the trial site, which could further explain the reasons for little differences. The DFM and YCW used alone or in combination cannot be expected to show productive benefits when animals are relatively unstressed with a low pathogenic bacterial challenge.

Prevalence, geographic distribution and risk factors of Eimeria species on commercial broiler farms in Guangdong, China.

BACKGROUND: Coccidiosis is one of the most frequently reported diseases in chickens, causing a significant economic impact on the poultry industry. However, there have been no previous studies evaluating the prevalence of this disease in broiler farms in Guangdong province. Therefore, this study aims to conduct an epidemiological investigation into the occurrence of Eimeria species and associated risk factors in intensive management conditions across four regions in Guangdong province, China. A total of 394 fecal samples were collected from 89 broiler farms in Guangdong province. The prevalence of Eimeria species infection was determined using PCR, and the occurrence of Clostridium perfringens type A was assessed using quantitative real-time PCR. RESULTS: The results showed an overall prevalence of 98.88% (88/89) at the farm level and 87.06% (343/394) at the flock level. All seven Eimeria species were identified, with E. acervulina (72.53%; 64/89), E. tenella (68.54%; 61/89), and E. mitis (66.29%; 59/89) at the farm level, and E. acervulina (36.55%; 144/394), E. mitis (35.28%; 139/394), and E. tenella (34.01%; 134/394) at the flock level. The predominant species combination observed was a co-infection of all seven Eimeria species (6.74%; 6/89), followed by a combination of E. acervulina, E. tenella, E. mitis, E. necatrix, E. brunetti, and E. maxima (5.62%, 5/89). A combination of E. acervulina, E. tenella, E. mitis, E. necatrix, E. brunetti, and E. praecox (4.49%; 4/89) was also observed at the farm level. Furthermore, the study identified several potential risk factors associated with the prevalence of Eimeria species, including farm location, chicken age, drinking water source, control strategy, and the presence of C. perfringens type A were identified as potential risk factors associated with prevalence of Eimeria species. Univariate and multivariate analyses revealed a significant association between E. necatrix infection and both grower chickens (OR = 10.86; 95% CI: 1.92-61.36; p < 0.05) and adult chickens (OR = 24.97; 95% CI: 4.29-145.15; p < 0.001) compared to starter chickens at the farm level. Additionally, farms that used groundwater (OR = 0.27; 95% CI: 0.08-0.94; p < 0.05) were less likely to have E. maxima compared to those that used running water. At the flock level, the prevalence of E. tenella was significantly higher in the Pearl River Delta (OR = 2.48; 95% CI: 1.0-6.15; p = 0.05) compared to eastern Guangdong. Interestingly, flocks with indigenous birds were less likely to have E. brunetti (OR = 0.48; 95% CI: 0.26-0.89; p < 0.05) compared to flocks with indigenous crossbred birds. Furthermore, flocks that used anticoccidial drugs (OR = 0.09; 95% CI: 0.03-0.31; p < 0.001) or a combination of vaccines and anticoccidial drugs (OR = 0.06; 95% CI: 0.01-0.25; p < 0.001) were less likely to be positive for E. tenella compared to flocks that only used vaccines. Finally, flocks with C. perfringens type A infection were significantly more likely to have E. necatrix (OR = 3.26; 95% CI: 1.96-5.43; p < 0.001), E. tenella (OR = 2.14; 95% CI: 1.36-3.36; p < 0.001), E. brunetti (OR = 2.48; 95% CI: 1.45-4.23; p < 0.001), and E. acervulina (OR = 2.62; 95% CI: 1.69-4.06; p < 0.001) compared to flocks without C. perfringens type A. CONCLUSIONS: This study conducted an investigation on the prevalence, distribution, and risk factors associated with Eimeria species infection in broiler chickens in Guangdong. The farm-level prevalence of Eimeria species was higher than the previous prevalence figures for other areas and countries. E. brunetti was identified at higher prevalence in Guangdong than previously survived prevalence in different regions in China. Farm location, chicken age, drinking water source, control strategy, and the presence of C. perfringens type A were considered as potential risk factors associated with prevalence of Eimeria species. It is imperative to underscore the necessity for further surveys to delve deeper into the occurrence of Eimeria species under intensive management conditions for different flock purposes.

Genetic diversity and phylogenetic relationships of Clostridium perfringens strains isolated from mastitis and enteritis in Egyptian dairy farms.

BACKGROUND: Clostridium perfringens, a common environmental bacterium, is responsible for a variety of serious illnesses including food poisoning, digestive disorders, and soft tissue infections. Mastitis in lactating cattle and sudden death losses in baby calves are major problems for producers raising calves on dairy farms. The pathogenicity of this bacterium is largely mediated by its production of various toxins. RESULTS: The study revealed that Among the examined lactating animals with a history of mastitis, diarrheal baby calves, and acute sudden death cases in calves, C. perfringens was isolated in 23.5% (93/395) of the total tested samples. Eighteen isolates were obtained from mastitic milk, 59 from rectal swabs, and 16 from the intestinal contents of dead calves. Most of the recovered C. perfringens isolates (95.6%) were identified as type A by molecular toxinotyping, except for four isolates from sudden death cases (type C). Notably, C. perfringens was recovered in 100% of sudden death cases compared with 32.9% of rectal swabs and 9% of milk samples. This study analyzed the phylogeny of C. perfringens using the plc region and identified the plc region in five Egyptian bovine isolates (milk and fecal origins). Importantly, this finding expands the known data on C. perfringens phospholipase C beyond reference strains in GenBank from various animal and environmental sources. CONCLUSION: Phylogenetic analyses of nucleotide sequence data differentiated between strains of different origins. The plc sequences of Egyptian C. perfringens strains acquired in the present study differed from those reported globally and constituted a distinct genetic ancestor.

Clostridium perfringens-induced liver abscess with severe haemolysis.

Clostridium perfringens is notorious for causing skin and soft tissue infections and food poisoning. Rarely, C. perfringens infections are associated with severe haemolysis, with a mortality rate of >80%. A previously healthy man in his 70s who presented with fever as his chief symptom was promptly admitted to a regional core hospital. Over the next 3 hours, shock and multiple organ failure ensued, leading to referral to our hospital for intensive care. We suspected a liver abscess caused by C. perfringens infection with haemolysis, findings of severe haemolysis and a liver mass with gas production that appeared within a few hours. Though surgical drainage was contemplated, low blood pressure resulted in death within 3 hours of arrival at our hospital. The next day, a blood culture confirmed C. perfringens, proving the diagnosis. Improving patient outcomes requires increased awareness of the disease and early detection.

An Upper-Arm Clostridium perfringens Fracture-Related Infection: A Case Report.

CONCLUSION: Fracture-related infections (FRI) pose serious complications, requiring swift surgical intervention. Although C. perfringens infections in FRIs are rare and literature is scarce, this case highlights the successful management and good functional outcome, offering valuable insights for clinicians dealing with such infections.

Extract of Scutellaria baicalensis and Lonicerae flos improves growth performance, antioxidant capacity, and intestinal barrier of yellow-feather broiler chickens against Clostridium perfringens.

In this study, we aimed to investigate the effect of Scutellaria baicalensis and Lonicerae Flos (SL) extract on the growth performance and intestinal health of yellow-feather broilers following a Clostridium perfringens challenge. In total, 600 one-day-old yellow-feather broilers were divided into five treatments (6 replicate pens of 20 birds per treatment), including a control (Con) group fed a basal diet and the infected group (iCon) fed a basal diet and infected with Clostridium perfringens, the other 3 groups receiving different doses of SL (150, 300, and 450 mg/kg) and infected with Clostridium perfringens. The total experimental period was 80 d. When the birds were 24-days-old, a subclinical necrotizing enteritis model was induced by orally inoculating the birds with 11,000 oocysts of mixed Eimeria species on d 24, followed by C. perfringens (108 CFU/mL) from d 28 to 30. The birds were evaluated for parameters such as average weight gain (AWG), average daily feed intake (ADFI), mortality, feed conversion ration (FCR), intestinal lesion score, intestinal C. perfringens counts, and villus histomorphometry. Results indicated that C. perfringens infection led to reduced AWG and the levels of tight junction proteins, increased the FCR, ileum E. coli load, and intestinal permeability, causing damage to the intestinal mucosal barrier (P < 0.05). Compared with the infected group, supplementing 300 mg/kg of SL significantly increased AWG at 43 to 80 d, the ratio of villus height to crypt depth in the jejunum and ileum at 35 d, and the activity of superoxide dismutase (SOD) in serum. It also significantly reduced the FCR at 22 to 42 d, intestinal lesion score, and the amount of C. perfringens in the ileum (P < 0.05). Additionally, compared with the infected group, the addition of 300 mg/kg SL significantly increased mRNA levels of claudin-2, claudin-3, mucin-2, and toll-like receptor 2 (TLR-2) in the ileum of infected birds at 35 d of age. In conclusion, supplementation with SL extract could effectively mitigate the negative effects of C. perfringens challenge by improving intestinal barrier function and histomorphology, positively influencing the growth performance of challenged birds.

Effect of arginine supplementation on the growth performance, intestinal health, and immune responses of broilers during necrotic enteritis challenge.

The objective of this study was to evaluate the effect of 25% arginine supplementation as a functional amino acid in partially alleviating the detrimental effects of necrotic enteritis (NE) on the growth performance, serum biochemistry, gut integrity, and the relative gene expression of tight junction proteins and inflammatory cytokines in broilers during NE. Three hundred and sixty 1-day-old chicks were randomly allocated to 4 treatments in a 2 × 2 factorial arrangement -basal diet and 125% arginine diet, with or without NE challenge. NE was induced by inoculating 1 × 104Eimeria maxima sporulated oocysts on d 14 and 1 × 108 CFU/bird C. perfringens on d 19, 20, and 21. The NE challenge had a significant effect on the BWG (p < 0.05), FCR (p < 0.05), serum AST (p < 0.05), GLU (p < 0.05), and K+ (p < 0.05) levels, and intestinal permeability (p < 0.05) and jejunal lesion score (p < 0.05). A significant challenge × diet interaction effect was observed in the cecal tonsil CD8+: CD4+ T-cell ratio on d 21 (p < 0.05) and 28 (p < 0.05) and spleen CD8+: CD4+ T-cell ratio on d 21 (p < 0.05) and 35 (p < 0.05). Arginine supplementation significantly increased the CD8+: CD4+ T-cell ratio in uninfected birds but decreased the CD8+: CD4+ T-cell ratio in infected birds. On d 21, a significant interaction effect was observed on the relative expression of the iNOS gene (p < 0.05). Arginine supplementation significantly downregulated the expression of the iNOS gene in infected birds. A significant effect of the challenge (p < 0.05) was observed on the relative gene expression of the ZO-1 gene in the jejunum. NE challenge significantly downregulated the expression of the ZO-1 gene on d 21. In conclusion, arginine supplementation did not alleviate the depression in growth performance and disease severity during the NE challenge. However, arginine downregulated the expression of inflammatory cytokines and enzymes, preventing inflammatory injury to the tissues during NE. Hence, arginine might be supplemented with other alternatives to downregulate inflammatory response during NE in poultry.

Effect of 125% and 135% arginine on the growth performance, intestinal health, and immune responses of broilers during necrotic enteritis challenge.

The objective of this study was to evaluate the effects of 25% and 35% arginine supplementation in partially alleviating the effects of necrotic enteritis (NE) challenge on the production performance, intestinal integrity, and relative gene expression of tight junction proteins and inflammatory cytokines in broilers. Four hundred and eighty 1-day-old chicks were randomly allocated to the 4 treatments- Uninfected + Basal, NE + Basal, NE + Arg 125%, and NE + Arg 135%. NE was induced by inoculating 1 × 104Eimeria maxima sporulated oocysts on d 14 and 1 × 108 CFU/bird C. perfringens on d 19, 20, and 21 of age by oral gavage. The NE challenge significantly decreased body weight gain (BWG) (p < 0.05) and increased the feed conversion ratio (FCR) (p < 0.05). On d 21, the NE challenge also increased the jejunal lesion score (p < 0.05) and relative gene expression of IL-10 and decreased the expression of the tight junction proteins occludin (p < 0.05) and claudin-4 (p < 0.05). The 125% arginine diet significantly increased intestinal permeability (p < 0.05) and the relative gene expression of iNOS (p < 0.05) and IFN-γ (p < 0.05) on d 21 and the bile anti-C. perfringens IgA concentration by 39.74% (p < 0.05) on d 28. The 135% arginine diet significantly increased the feed intake during d 0 - 28 (p < 0.05) and 0 to 35 (p < 0.05) and increased the FCR on d 0 to 35 (p < 0.05). The 135% and 125% arginine diet increased the spleen CD8+: CD4+ T-cell ratio on d 28 (p < 0.05) and 35 (p < 0.05), respectively. The 135% arginine diet increased the CT CD8+:CD4+ T-cell ratio on d 35 (p < 0.05). In conclusion, the 125% and 135% arginine diets did not reverse the effect of the NE challenge on the growth performance. However, the 125% arginine diet significantly increased the cellular and humoral immune response to the challenge. Hence, the 125% arginine diet could be used with other feed additives to improve the immune response of the broilers during the NE challenge.

Supplementation with organic yeast-derived selenium provides immune protection against experimental necrotic enteritis in broiler chickens.

Necrotic enteritis (NE) is a potentially fatal poultry disease that causes enormous economic losses in the poultry industry worldwide. The study aimed to evaluate the effects of dietary organic yeast-derived selenium (Se) on immune protection against experimental necrotic enteritis (NE) in commercial broilers. Chickens were fed basal diets supplemented with different Se levels (0.25, 0.50, and 1.00 Se mg/kg). To induce NE, Clostridium perfringens (C. perfringens) was orally administered at 14 days of age post hatch. The results showed that birds fed 0.25 Se mg/kg exhibited significantly increased body weight gain compared with the non-supplemented/infected birds. There were no significant differences in gut lesions between the Se-supplemented groups and the non-supplemented group. The antibody levels against α-toxin and NetB toxin increased with the increase between 0.25 Se mg/kg and 0.50 Se mg/kg. In the jejunal scrapings and spleen, the Se-supplementation groups up-regulated the transcripts for pro-inflammatory cytokines IL-1ß, IL-6, IL-8, iNOS, and LITAF and avian ß-defensin 6, 8, and 13 (AvBD6, 8 and 13). In conclusion, supplementation with organic yeast-derived Se alleviates the negative consequences and provides beneficial protection against experimental NE.

Genomic analysis of Clostridium perfringens type D isolates from goat farms.

C. perfringens type D strains are the leading cause of enterotoxaemia in ruminants such as goats, sheep, and cattle. However, there has been no prior research on the genomic characteristics of C. perfringens type D strains from various regions in China. Here, we investigated the antibiotic resistance, genomic characteristics, and phylogenetic relationship of C. perfringens type D isolates recovered from goat farms in Shaanxi, Gansu, and Ningxia provinces. The antibiotic resistance test indicated that the isolates displayed high minimum inhibitory concentration (MIC) values to sulfafurazole, whereas the other antibiotics tested, such as penicillin, enrofloxacin, and florfenicol, worked well on them. Additionally, only tetracycline resistance genes [tetA(P) and tetB(P)] were identified from the isolates. A collective of 13 toxin genes, including etx and cpe were detected among the isolates. Sequence comparison revealed that the etx and cpe genes shared high sequence identities, and they could coexist on a pCW3-like plasmid, representing a potential risk to both animal breeding and public health. Phylogenetic analysis using core genome multi-locus sequence typing (cgMLST) and core genome single nucleotide polymorphisms (SNPs) revealed the close genetic relationship and potential regional/transregional transmission of the C. perfringens type D isolates in Shaanxi and Gansu provinces. Furthermore, pan-genomic analysis suggested the functional differences at the protein-coding gene level, although isolates from the same source shared a close genetic relationship. In conclusion, this study indicated the antibiotic resistance, virulence markers, potential transregional transmission, and genomic diversity of C. perfringens type D strains from various regions in China, which could provide references for the prevention of C. perfringens foodborne diseases and further research.

Identification and structure-based engineering of a dipeptidase CpPepD from Clostridium perfringens for the synthesis of l-carnosine.

l-Carnosine (l-Car), an endogenous dipeptide presents in muscle and brain tissues of various vertebrates, has a wide range of application values. The enzymatic preparation of l-Car is a promising synthetic method because it avoids the protection and deprotection steps. In the present study, a dipeptidase gene (CpPepD) from Clostridium perfringens with high l-Car synthetic activity was cloned and characterized. In an effort to improve the performance of this enzyme, we carried out site saturation mutagenesis using CpPepD as the template. By the o-phthalaldehyde (OPA)-derived high throughput screening method, mutant A171S was obtained with 2.2-fold enhanced synthetic activity. The enzymatic properties of CpPepD and mutant A171S were investigated. Under the optimized conditions, 63.94 mM (14.46 g L-1) or 67.02 mM (15.16 g L-1) l-Car was produced at the substrate concentrations of 6 M ß-Ala and 0.2 M l-His using wild-type or mutant A171S enzyme, respectively. Although the mutation enhanced the enzyme activity, the reaction equilibrium was barely affected.

Intestinal Colonization by Campylobacter jejuni, Clostridium difficile, and Clostridium perfringens among Commensal Rattus norvegicus in the Urban Areas of Tehran, Iran.

Background: Rattus norvegicus (R. norvegicus) population plays a significant role in the spread of numerous diseases in urban environments. The present study is aimed at investigating the presence of Campylobacter jejuni (C. jejuni), C. coli, Clostridium difficile (C. difficile), C. difficile toxigenic, and C. perfringens in R. norvegicus captured from urban areas of Tehran, Iran. Methods: From October 2021 to October 2022, 100 urban rats were trapped in 5 different districts of Tehran, Iran. The genomic DNA was extracted from fecal samples, and the presence of C. jejuni, C. coli, C. perfringens, and C. difficile species was evaluated using PCR assay. Moreover, PCR was used to assess the toxicity of C. difficile isolates. Results: Overall, 30% (n = 30/100) of fecal samples were positive for zoonotic pathogens. Based on the PCR on hippuricase (hipO), glycine (gly), CIDIF, and phospholipase C (plc) genes, C. perfringens and C. difficile were isolated from 18.2% (n = 14/77) and 5.2% (n = 4/77) of male rats. The highest frequency of C. perfringens and C. jejuni was 25% (n = 5/20) related to the south of Tehran. Toxigenic C. difficile was not detected in all regions. Conclusion: According to the findings, rats are the main reservoirs for diseases. Therefore, rodent control coupled with the implementation of surveillance systems should be prioritized for urban health.

Injury of Macrophages Induced by Clostridium perfringens Type C Exotoxins.

Clostridium perfringens is a kind of anaerobic Gram-positive bacterium that widely exists in the intestinal tissue of humans and animals. And the main virulence factor in Clostridium perfringens is its exotoxins. Clostridium perfringens type C is the main strain of livestock disease, its exotoxins can induce necrotizing enteritis and enterotoxemia, which lead to the reduction in feed conversion, and a serious impact on breeding production performance. Our study found that treatment with exotoxins reduced cell viability and triggered intracellular reactive oxygen species (ROS) in human mononuclear leukemia cells (THP-1) cells. Through transcriptome sequencing analysis, we found that the levels of related proteins such as heme oxygenase 1 (HO-1) and ferroptosis signaling pathway increased significantly after treatment with exotoxins. To investigate whether ferroptosis occurred after exotoxin treatment in macrophages, we confirmed that the protein expression levels of antioxidant factors glutathione peroxidase 4/ferroptosis-suppressor-protein 1/the cystine/glutamate antiporter solute carrier family 7 member 11 (GPX4/FSP1/xCT), ferroptosis-related protein nuclear receptor coactivator 4/transferrin/transferrin receptor (NCOA4/TF/TFR)/ferritin and the level of lipid peroxidation were significantly changed. Based on the above results, our study suggested that Clostridium perfringens type C exotoxins can induce macrophage injury through oxidative stress and ferroptosis.

The Effects of Encapsulation on the In Vitro Anti-Clostridial Activity of Olive Mill Wastewater Polyphenolic Extracts: A Promising Strategy to Limit Microbial Growth in Food Systems.

Despite the technologies applied to food production, microbial contamination and chemical deterioration are still matters of great concern. In order to limit these phenomena, new natural approaches should be applied. In this context, the present study aimed to assess the antioxidant and anti-Clostridial effects of two different polyphenolic extracts derived from olive mill vegetation water, one liquid (LE) and one encapsulated (EE). The extracts have been preliminary characterized using Liquid Chromatography Quadrupole Time-Of Flight spectrometry. The Oxygen Radical Absorbance Capacity method was used to determine the antioxidant capacity, registering a higher value for EE compared to that for LE (3256 ± 85 and 2446 ± 13 µgTE/g, respectively). The antibacterial activity against C. perfringens, C. botulinum and C. difficile was studied by the agar well diffusion method, MIC and MBC determination and a time-kill test. The results confirm that EE and LE are able to limit microbial growth, albeit with minor effects when the phenolic compounds are encapsulated. Further studies are needed to evaluate the possible application of these extracts in food systems.

An in vitro assay for toxicity testing of Clostridium perfringens type C ß-toxin.

Introduction: Veterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the ß-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the ß-toxin. Methods: Development of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for ß-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout. Results: The current animal test is estimated to detect 100 - 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the ß-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability. Conclusions: Here, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay's sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control.

Short communication: a novel multispecies bacteria-based direct-fed microbial supports in vitro gut barrier integrity challenged with a pathogen or pro-inflammatory cytokines.

We conducted two experiments to evaluate the effects of a novel bacterial-based direct-fed microbial (DFM) on intestinal barrier integrity using the in vitro transepithelial electrical resistance (TEER) assay. In experiment 1, human-derived Caco-2 cells received or not (CON) a DFM containing Ligilactobacillus (formerly Lactobacillus) animalis 506, Propionibacterium freudenreichii 507, Bacillus paralicheniformis 809, and B. subtilis 597 (BDP; BOVAMINE DEFEND® Plus) at a rate of 1 × 108 CFU/transwell. Concurrently with treatment application (CON or BDP), a pathogenic challenge of Clostridium perfringens type A was added alone (PAT) or with BDP (PAT + BDP) at a rate of 2.8 × 107 CFU/transwell in a 2 × 2 factorial arrangement. In experiment 2, Caco-2 cells were also assigned in a 2 × 2 factorial design to CON or BDP and then, 2 h post-treatment administration (CON and BDP), a mixture of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) was added alone (CYT) or with BDP (CYT + BDP) at a 10:1 ratio, respectively. In both experiments, TEER was measured for 18 h. In experiment 1, a DFM × pathogen × hour interaction was observed for TEER (P < 0.0001). Adding the PAT alone initially tended to increase TEER vs. CON from 1.1 to 2.2 h (P ≤ 0.09), increased TEER at 3.2 h (P < 0.01), but reduced TEER from 5.4 to the end of the experimental period at 18.4 h (P ≤ 0.01). On the other hand, adding DFM, with or without the pathogenic challenge, yielded greater TEER vs. CON-CON and CON-PAT for most of the experimental period (P ≤ 0.04). A similar interaction was detected and reported in experiment 2 (P < 0.0001). The CYT challenge reduced mean TEER compared with all other treatments from 3.2 h to the remainder of the study (P ≤ 0.03). On the other hand, BDP-CYT was able to maintain the integrity of the epithelial cells when compared with CON-CON throughout the experimental period (P ≤ 0.03), the exception being at 3.2 h (P = 0.20). Moreover, BDP-CON increased (P ≤ 0.04) TEER when compared with CON-CON from 3.2 to 18.4 h, but also in comparison with BDP-CYT from 4.3 to 18.4 h post-DFM and challenge administration into the cells. In summary, C. perfringens type A and a pro-inflammatory cytokine cocktail compromised the integrity of intestinal epithelial cell monolayers in vitro, whereas adding a multispecies bacteria-based DFM counteracted these damaging effects.

Two experiments were designed to evaluate the effects of adding a bacterial-based direct-fed microbial (DFM) containing Lactobacillus animalis 506, Propionibacterium freudenreichii 507, Bacillus paralicheniformis 809, and Bacillus subtilis 597 on the integrity of intestinal epithelial cells challenged with Clostridium perfringens type A or a pro-inflammatory cytokine cocktail. Regardless of the challenge, the addition of the DFM maintained the integrity of the intestinal epithelial cells in vitro. These results help to elucidate the potential beneficial effects that the bacterial-based DFM containing L. animalis 506, P. freudenreichii 507, B. paralicheniformis 809, and B. subtilis 597 may bring to livestock species.

Hematological investigations in a case of intravascular hemolysis due to Clostridium perfringens infection.

A patient presented with fever, severe pain and edematous tight due to hip trauma and was scheduled for urgent fasciotomy. Following physical examination, laboratory analyses were requested, and results revealed anemia and severe infection. As the patient's condition was serious, a new set of samples was sent to the laboratory four hours later. Following centrifugation, severely hemolyzed dark-colored serum and plasma samples were obtained and in vitro hemolysis was suspected. The collection of samples was repeated, but a new set of samples was also hemolyzed with a significant decrease in the hemoglobin value. At that point, in vivo hemolysis was suspected, and samples were processed according to standard laboratory procedures for hemolytic samples. Following confirmation of the gas gangrene diagnosis by clinicians, the cause of hemolysis was attributed to the cytotoxic activity of α-toxin produced by the anaerobic gram-positive bacterium Clostridium perfringens. An insight into the laboratory procedure that could help to narrow down the causes of hemolysis and single out C. perfringens as a cause of intravascular hemolysis was given.

The Molecular Architecture and Mode of Action of Clostridium perfringens ε-Toxin.

Clostridium perfringens ε-toxin has long been associated with a severe enterotoxaemia of livestock animals, and more recently, was proposed to play a role in the etiology of multiple sclerosis in humans. The remarkable potency of the toxin has intrigued researchers for many decades, who suggested that this indicated an enzymatic mode of action. Recently, there have been major breakthroughs by finding that it is a pore-forming toxin which shows exquisite specificity for cells bearing the myelin and lymphocyte protein (MAL) receptor. This review details the molecular structures of the toxin, the evidence which identifies MAL as the receptor and the possible roles of other cell membrane components in toxin binding. The information on structure and mode of action has allowed the functions of individual amino acids to be investigated and has led to the creation of mutants with reduced toxicity that could serve as vaccines. In spite of this progress, there are still a number of key questions around the mode of action of the toxin which need to be further investigated.